There has been rapid growth in recent years in apparatus and methodology for biochemical enterprise, particularly in the development of increasingly sophisticated systems for automating biochemical processes.
Procedures in chemistry, particularly in biochemistry, present generally more difficult problems for automation than many other kinds of processes and procedures. One reason is that there is often a very long sequence of steps in a biochemical procedure, such as gene detection and sequencing DNA. Another is that an automatic system needs to be very versatile, because different kinds of starting materials and different analytical purposes require different steps, different order of steps and the use of different kinds of chemical reagents. A third is that sample quantity is, for various reasons, quite limited, and only very small volumes, often on the order of microliters, must be used.
Systems have been designed to accomplish procedures useful in biochemical analysis, such as transfer of liquid from one container to another by pipette, and in general such systems mimic manual procedures. Typically a mechanical arm is moved over a limited area and carries one or more pipette tips. Systems of the prior art, however, have been mostly addressed to protocols in which liquid transfer is in volumes much larger then the microliter volumes often encountered in biochemical procedures, and these systems have been less than notably successful in addressing problems created by conditions such as those described above, like pipetting very small quantities of liquid with accuracy.
Aspirating liquid into and dispensing liquid from a pipette can be done several different ways. If a liquid is dispensed into air relatively rapidly, the liquid is dispensed at a regular rate, that is, in an analog fashion. If the same liquid is dispensed relatively slowly, the dispensing rate becomes, at some point, incremental. A droplet forms on the tip, grows, and separates from the tip, then another droplet grows and separates. The size of the droplet depends on such variables as the diameters and the design of the tip and the viscosity and surface tension of the liquid being dispensed. The viscosity and surface tension depend on other variables, among them the liquid material and the temperature.
The droplet phenomenon affects aspiration of liquid into a pipette from a container of liquid as well. Liquid is aspirated with a pipette below the surface of liquid in a container, but when the tip is withdrawn, a droplet can form on the tip, and affect the accuracy of the aspiration. The effect of the droplet size on accuracy depends on the volume to be aspirated and the droplet volume.
If a volume to be aspirated or dispensed is very large compared to the droplet size that forms on the pipette tip, the droplet phenomenon has little effect on accuracy. If, however, the amount to be aspirated or dispensed is in the range of, for example, ten times the volume of a single drop, the droplet phenomenon can be serious indeed, and accuracy may be seriously impaired. In the case of biochemical procedures, the sample size and the volume of material to be aspirated and dispensed is typically very small. If a liquid to be handled is quite viscous, such as genomic DNA for example, the droplet problem assumes larger proportions.
If liquid is to be dispensed into a container, and the container already contains liquid, the pipette tip can be submerged in the liquid in the container, much in the manner that liquid is typically aspirated, then additional liquid may be dispensed in an analog fashion. A new problem in this procedure, however, is that when the pipette is withdrawn from the liquid in the container, an uncontrolled amount of the liquid can adhere to the outside of the pipette and be carried away when the pipette is moved. Again, if the volume to be aspirated is large compared to the amount that adheres to the pipette, the inaccuracy is small. If the amount to be aspirated, however is small, as is typically the case in biochemical procedures, such as DNA sequencing, the amount that adheres to the outside of the pipette may introduce significant error. Also, the further a tip is immersed in a liquid whether aspirating or dispensing, the more liquid can adhere to the tip, and the greater may be the inaccuracy.
Another problem encountered is with the speed and precision of robotics. A robot for moving a pipette to accomplish liquid transfers from container to container is in some respects a simpler problem than manipulating solid objects. For example, a robot to do pipetting requires three degrees of freedom, while some robot devices require as many as seven. In biochemical procedures, however, it is generally necessary to access a large number of different sites, and to do so very accurately. It is desirable in gene detection and DNA sequencing, for example, to process a relatively large number of samples in a single procedure. To do so requires the addition of many different reagents for each sample, and the needed reagents are not in every case the same for each sample. There have to be sites in the scanned area of the robot arm for containers to hold all of the samples and for all of the necessary liquids to perform the procedures. Moreover, there is a need for other sites, such as a wash station for the pipette or pipettes and stations for such procedures as mixing, incubating, separating, and the like.
In the case of biochemical procedures, the number of sites and the lengthy procedures require that movement from site to site be accomplished quickly to save time. Moreover, the requirement for small volumes of samples and other liquids imposes a restriction of small containers, hence small targets for the pipette. Accuracy and resolution become more important for small targets.
Systems of the prior art mimic the manual processes of pipetting very poorly. A laboratory worker using a manual pipette develops detailed technique for pipetting liquids, and often employs such technique without considerable thought. For example, a worker will typically develop technique for approaching the surface of a liquid with a pipette tip very slowly, and will move the tip slowly and with precision at the liquid surface. A worker will also typically employ technique such as touching a droplet on the pipette to the surface of a liquid to transfer the droplet to the liquid mass. These movements made almost without conscious thought by a skilled worker are difficult to duplicate with a robot, and are typically not accomplished in automatic systems of the prior art.
Yet another problem encountered in automating biochemical procedures such as gene detection and DNA sequencing is associated with the systems of programming and control. It is known to operate such systems with computers and to program sequences of action for a computer to follow to accomplish the chemical procedures, but the large variation in steps, variation in variables such as heating, cooling and mixing, and the need to process a large number of samples at a time imposes a severe requirement for a system that is flexible and operator friendly, with an operator interface that is easy to use to set up process variations.
Still another problem encountered in the design of such a system is liquid integrity. Even with rapid movement of robotic components and short and compact site design, the large number of samples and large number of steps for each sample, coupled with time required for such things as heating and cooling, dictates that operations must be done over long periods, such as several hours. Given long processing times and small samples, evaporation can be a serious problem, and can cause significant uncontrolled changes in liquid concentration, introducing error. Moreover, open containers invite problems in cross-contamination. Such contamination can be from carryover with pipette operation and also from evaporation and condensation.
Another very serious problem with apparatus of the prior art is that such apparatus typically uses throw-away pipette tips, with a new tip being used for every pipette transfer. Such a system has to provide for discarding tips after use, a waste container to receive the discarded tips, storage for a large supply of fresh tips for use, and apparatus and control schemes for making the tip changes between liquid transfers. The apparatus and extra motions result in greater error than would result if a single tip could be used. Moreover, the need for discarding a tip and loading a new tip for each liquid transfer is time consuming, making the overall processing time more than would be necessary if a single tip could be used.
What is needed is automatic robotic apparatus for doing liquid transfers with very small quantities of liquids, and in a manner that avoids carryover and evaporation. Such an instrument needs to be modular in nature so that container stations may be interchanged, with modular stations for holding containers so that such operations as sample preparation and cleaning may be done off-line. Methods for operation of such apparatus are needed allowing a relatively large number of samples to be processed at a time, with samples and reagents placed in a close array to preserve space. The robotic actions need to be rapid to minimize overall processing time and extremely accurate to be able to access many small sites. Such a system also must incorporate robotic techniques to approximate human handling of pipette tips to accomplish adequate accuracy when operating with very small volumes of samples and reagents, and also when handling viscous liquids. The apparatus needs to provide a single pipette tip that can be reused to avoid the clumsy, time-consuming, and error-prone process of frequently discarding a tip and loading a new tip, and the problems of cross-contamination caused by single tip use must be addressed. The apparatus and associated methods of operation also must minimize evaporation and cross-contamination. Such an apparatus needs to be integrated with a control system that allows an operator to easily and quickly set up procedures with different variables, different step sequences, and different samples and reagents.
Also needed is laboratory apparatus based on such a liquid handling system to incorporate further techniques, such as temperature control and a separation station, to be able to fully automate specific chemistry protocols such as for gene detection and DNA sample purification.